The invention is directed to a method for the synthesis of nucleotide derivatives wherein molecules of interest are grafted on the oligonucleotide with the help of a “click chemistry” reaction between an azide function on the molecule of interest and an alkyne function on the oligonucleotide, or between an alkyne function on the molecule of interest and an azide function on the oligonucleotide.
Other objects of the invention are intermediate molecules, notably alkyne functionalized oligonucleotides, azide functionalized oligonucleotides, grafted oligonucleotides, oligonucleotide micro arrays containing them and the use of those grafted oligonucleotides for biological investigation and for cell targeting among others.
Oligonucleotides are molecules consisting of a short chain of nucleotides, the number of which can vary from one to around one hundred. They are important molecular tools for genomic research and biotechnology (Caruthers, M. H. Science 1985, 230, 281-285). Most applications require labeling with dyes or other biomolecules such as peptides (Zatsepin, T. S.; Turner, J. J.; Oretskaya, T. S.; Gait, M. J. Curr. Pharm. Des. 2005, 11, 3639-3654), or carbohydrates (Zatsepin, T. S.; Oretskaya, T. S. Chem. Biodiversity 2004, 1, 1401-1417).
Oligonucleotides are typically synthesized on solid support using phosphoramidite chemistry (Beaucage, S. L.; Caruthers, M. H. Tetrahedron Lett. 1981, 22, 1859-1862). Their conjugation with carbohydrates has been performed on one hand on solid support using either solid-supported carbohydrates (Adinolfi, M.; De Napoli, L.; Di Fabio, G.; Iadonisi, A.; Montesarchio, D.; Piccialli, G. Tetrahedron 2002, 58, 6697-6704; D'Onofrio, J.; de Champdore, M.; De Napoli, L.; Montesarchio, D.; Di Fabio, G. Bioconjugate Chem. 2005, 16, 1299-1309) or carbohydrate phosphoramidites (Adinolfi, M.; De Napoli, L.; Di Fabio, G.; Iadonisi, A.; Montesarchio, D.; Piccialli, G. Tetrahedron 2002, 58, 6697-6704; D'Onofrio, J.; de Champdore, M.; De Napoli, L.; Montesarchio, D.; Di Fabio, G. Bioconjugate Chem. 2005, 16, 1299-1309; Sheppard, T. L.; Wong, C. H.; Joyce, G. F. Angew. Chem., Int. Ed. 2000, 39, 3660-3663; Tona, R.; Bertolini, R.; Hunziker, J. Org. Lett. 2000, 2, 1693-1696; de Kort, M.; de Visser, P. C.; Kurzeck, J.; Meeuwenoord, N. J.; van der Marel, G. A.; Rüger, W.; van Boom, J. H. Eur. J. Org. Chem. 2001, 2075-2082; Dubber, M.; Frechet, J. M. J. Bioconjugate Chem. 2003, 14, 239-246), and on the other hand in solution using reactive carbohydrate derivatives (Akasaka, T.; Matsuura, K.; Emi, N.; Kobayashi, K. Biochem. Biophys. Res. Commun. 1999, 260, 323-328; Forget, D.; Renaudet, O.; Defrancq, E.; Dumy, P. Tetrahedron Lett. 2001, 42, 7829-7832; Dey, S.; Sheppard, T. L. Org. Lett. 2001, 3, 3983-3986). Nevertheless, these strategies require multi-step synthesis and are time consuming.
There remained the need for a method permitting the grafting of varied molecules of interest on an oligonucleotide backbone, with the possibility to graft several different molecules of interest on the oligonucleotide backbone, wherein said method permits the grafting of molecules of interest either on the 3′-, or on the 5′-extremity of the oligonucleotide, or inside the sequence.
The inventors have found that the use of a “click chemistry” reaction between an azide function on the molecule of interest and an alkyne function grafted on a phosphonate diester functionalization of the oligonucleotide permitted to reach this goal.